This invention relates generally to antibodies which specifically bind to Hepatitis E Virus (HEV), and more particularly, relates to a novel hybridoma cell line which secretes monoclonal antibodies to HEV off-3 antigen, and methods for using these monoclonal antibodies.
HEV, variously referred to as waterborne, epidemic or enterically transmitted non-A, non-B hepatitis (ET-NANBH), has a global distribution and has been noted as the cause of major endemic outbreaks of viral hepatitis in developing countries. D. W. Bradley et al., Br. Med. Bull. 46:442-461 (1990). Sporadic cases of ET-NANBH, as well as imported travel exposure, have been reported in developed countries. S. J. Skidmore et al., Lancet 337:1541 (1991). Although the fecal-oral route of transmission predominates, limited person-to-person routes of exposure have been suggested in some epidemiological studies. O. Velasquez et al., J. Amer. Med. Assoc. 363:3281-3285 (1990). This disease has been documented as having a high mortality rate of approximately 20% in pregnant women infected during their third trimester of pregnancy. See D. W. Bradley et al., supra.
Molecular cloning of the putative agent of HEV has been hampered by the lack of a tissue culture system for virus propagation. However, the use of available animal models and a newly developed non-specific amplification procedure have allowed the identification of a unique cDNA clone (identified as "ET 1.1") obtained from bile of cynomolgus macaques infected with a Burmese strain of HEV. A. G. Andjaparidze et al., Vopr. Virusol. 1:73-80 (1986), D. W. Bradley et al., Proc. Natl. Acad. Sci. USA 84:6277-6281 (1987) and G. W. Reyes et al., Science 247:1335-1339 (1990). Successful confirmation of the viral origin of this clone led to the identification of similar sequences in human fecal specimens collected from ET-NANBH epidemics in Somalia, Tashkent, Borneo, Pakistan and Mexico. G. R. Reyes et al., supra. cDNA libraries also have been prepared from human stool samples obtained during an ET-NANBH outbreak in Mexico. G. R. Reyes et al., Gastroenterol. Japon. 26:142-147 (1991). Immunoscreening of these cDNA libraries led to the identification of two cDNA clones which encode epitopes specific for HEV. P. O. Yarbough et al., J. Virol. 65:5790-5797 (1991). The isolation and sequencing of a set of overlapping cDNA clones led to the recognition that this form of hepatitis is caused by a novel virus unlike any of the other molecularly characterized agents of viral hepatitis. A. W. Tam et al., Virology 185:12-131 (1991).
Various regions of the HEV genome have been cloned and expressed in E. coli as fusion proteins with glutathione-S-transferase (GST). See, for example, S. J. Skidmore et al., supra. Four of these recombinant antigens, two derived from a Burmese (B) strain of HEV and two derived from a Mexican (M) strain of HEV, have been shown to contain antigenic sites recognized by antibodies from individuals previously exposed to HEV. See, P. O. Yarbough et al., supra. The two antigens from the Mexican strain, named M3-2 and M4-2, correspond to amino acid sequences at the carboxy-terminus of the second open reading frame (ORF-2) and the third open reading frame (orf-3), respectively. The two antigens from the Burmese strain, B 3-2 and B 4-2, correspond to amino acid sequences at the carboxy-terminus of orf-2 and orf-3, respectively. The M 3-2 and B 3-2 recombinant antigens both comprise 42 amino acids from the carboxy terminus of ORF-2. The degree of amino acid homology between these sequences of 42 amino acids is 90.5%. Id. The M 4-2 and B 4-2 recombinant antigens each comprise 33 amino acids from the carboxy terminus of orf-3; the degree of homology between these two sequences of 33 amino acids is 73.5%. Id.
Tests developed for detection of HEV must contain reagents which are useful for determining the specific presence of the virus in a test sample. The need therefore exists for reagents, such as monoclonal antibodies, capable of reacting only with HEV. Additionally, the ability to produce pure, specific monospecific antibodies is clearly of great importance for accurate identification, characterization, and purification of the HEV antigen.
While methods are available to confirm screening assay results for agents such as HIV, these techniques are not yet available for confirming the presence of HEV. Methods such as culturing HEV in vitro and a viral-based Western blot test are not available. While the detection of HEV nucleic acid may be done by performing PCR, this technique is tedious and expensive, requires special equipment such as a thermocycler, and turn-around time is up to 24 hours. Immunoelectron microscopy (IEM) has been used to confirm the presence of anti-HEV antibody, but the use of IEM is cost prohibitive as a routine confirmatory tool.
It therefore would be advantageous to provide a monoclonal antibody which can be used in accurate, rapid and cost effective methods for screening for HEV antigens or HEV antibodies in a test sample.